Process for removal of particle-forming proteins from blood sera

ABSTRACT

A process is provided for removal of particle-forming proteins from blood serum by extracting the serum with a halo-genated straight chain hydrocarbon solvent at a pH of within the range of from about 5.2 to about 5.6.

United States Patent Hagan et al.

[451 Dec. 1 9 1972 PROCESS FOR REMOVAL OF PARTICLE-FORMING PROTEINS FROM BLOOD SERA E. R. s uibaa'sons'j'la; new FkJiY' Filed: March 25, 1971 Appl. No.: 128,152

Assignee:

US. Cl ..2l0/2l, ZIO/DIG 23, 210/54, v 260/112, 424/11 Int. Cl. ..B0ld 12/00 [58] Iiield of Search 0/21, 54;424/l 1; 260/112 References Cited FOREIGN PATENTS OR APPLICATIONS 7/1970 Great Britain Primary ExaminerMichael Rogers Att0rneyLawrence S. Levinson, Merle J. -Smith, Donald J. Perrella [5 7 ABSTRACT A process is provided for removal of particle-forming proteins from blood serum by extracting the serum with a halo-genated straight chain hydrocarbon solvent at a pH of within the range of from about 5.2 to about 5.6.

8 Claims, N0 Drawings PROCESS FOR REMOVAL OF PARTICLE- FORMING PROTEINS FROM BLOOD SERA This invention relates to a process for removing particle-forming substances (e.g., proteins) from blood sera.

Blood sera for grouping and typing purposes must be pure and substantially free of particle-forming substances. Formation of proteinaceous particles in blood serum, withor without fibrinogen, occurs when'the serum is shaken, for example, for 2 to 5 hours. Several methods have been suggested for removing particleforming proteins from sera. For example, British Pat. No. 1,196,636 discloses a process for preparing pure serum albumins from normal and hemolyzed sera and plasma by precipitating undesirable proteins and dyestuffs contained in a solution of raw albumins by heating a 5-8 percent solution of raw albumin together with percent by volume of chloroform at a pH of 4.95-5.05 to a temperatureof 50 C.

Furthermore, serum albumin of standard quality can be obtained by a method of ethanol fractionation as described by Cohn et al., J. Am. Chem. Soc., 68, 3386, (1946)and 72,465,(l950).

It has now been found that the formation of proteinaceous particles in blood sera, for example in grouping and Rh typing serum, induced by shaking, can be substantially prevented or retarded by extracting the blood serum with a halogenated straight chain hydrocarbon e.g., chloroform, without the need for employing elevated temperatures, by carrying out the extraction at a pH of within the range of from about 5.2 to about 5 .6.

Thus, in accordance with the present invention, a process is provided for substantially preventing or retarding the formation of proteinaceous particles in blood serum induced by shaking, by removal of particle-forming proteins therefrom, which comprises adjusting pH of the serum to within the range of from about 5.2 to about 5.6 and preferably from about 5.2 to about 5.4, mixing the blood serum with a halogenated straight chain hydrocarbon, thereby causing the formation of a proteinaceous precipitate, and separating the precipitate from the serum.

In carrying out the process of the invention, the halogenated straight chain hydrocarbon in employed in a volume ratio to the serum of within the range of from about 1:2 to about 2:1 or greater; however, no apparent benefit is obtained employing ratios greater than 5:1.

The pH of the serum can be adjusted to the required level before or after the addition of the halogenated straight chain hydrocarbon thereto by admixing with dilute acid such as a mineral acid, for example hydrochloric acid, sulfuric acid or nitric acid, or an organic acid such as formic acid or acetic acid.

If desired, the blood serum once adjusted to a pH within the range of from about 5.2 to about 5.6, can be shaken to induce precipitation of proteinaceous particles prior to adding the halogenated straight chain hydrocarbon thereto.

The extraction is usually carried out at ambient temperature, although, if desired, temperatures ranging from about to about 35 C can be employed to expedite formation of the proteinaceous precipitate.

Upon mixing of the hydrocarbon solvent with the serum having a pH within the above defined range the mixture is shaken or mixed for a period ranging from about A to about 1 k hours to ensure complete admixture. Longer mixing periods can be employed, with no apparent benefit.

After mixing, the mixture is allowed to settle preferably at reduced temperatures ranging from about 0 to about 20 C, for periods ranging from about 1 to about 20 hours. The precipitate formed in the mixture can then be separated, for example, by centrifugation or other conventional technique.

The clear serum layer can then be removed and further purified, for example, by placing the serum under vacuum at a temperature within the range of from about 20 to about 40 C with stirring until foaming ceases, adjusting the pH of serum to within the range of from about 6.5 to about 8, for example by adding alkali thereto, such as sodium hydroxide, potassium hydroxide, sodium carbonate, ammonium hydroxide or tris(hydroxymethyl)aminomethane. Optionally, the serum can then be treated with an absorbent such as'kaolin, further centrifuged and filtered to further purify the serum.

The halogenated straight chain hydrocarbon solvents which can be employed herein include chloroform, methylene chloride or carbon tetrachloride.

The following examples represent preferred embodiments of the invention.

EXAMPLE 1 The pH or human serum (plasma with substantially all fibrinogen removed therefrom) is adjusted to a value of 5.2 by adding about 1.2 ml of dilute acetic acid (30 percent), to ml of serum. An equal volume of methylene chloride is added to the serum and the mixture is shaken for three hours at 30 C. The mixture is allowed to stand at room temperature for several hours and then is centrifuged at 5,000 X g for 10 minutes whereupon three layers form, a solvent layer, a layer containing a solid precipitate and a serum liquid layer. The three layers are separated and the serum layer is placed under vacuum at 35 with stirring (for at least 15 minutes) until foaming ceases. The pH of the serum is adjusted. to 7.2 by adding 5N sodium hydroxide thereto. The serum is then filtered through a sterile D-8 filter pad to thereby remove additional impurities therefrom.

EXAMPLE 2 The pH of human serum (plasma with substantially all fibrinogen removed therefrom) is adjusted to a value of 5.5 by adding about 0.9 ml of dilute acetic acid (30 percent), to 100 ml of serum. An equal volume of chloroform is added to the serum and the mixture is shaken for 5 hours at 45 C. The mixture is allowed to stand at room temperature for several hours and then is centrifuged at 10,000 X g for 10 minutes whereupon three layers form, a solvent layer, a layer containing a solid precipitate and a serum liquid layer. The three layers are separated and the serum layer is placed under vacuum at 35 with stirring (for at least 15 minutes) until foaming ceases. The pH of the serum is adjusted to 6.5-8 by adding 5N sodium hydroxide thereto. The serum is then filtered through a sterile D-8 filter pad to thereby removed additional impurities therefrom.

EXAMPLE 3 The pH of human serum (plasma with substantially all fibrinogen removed therefrom) is adjusted to a value of 5.4 by adding about 1 ml of dilute acetic acid (30 percent), to 100 ml of serum. About 200 ml of chloroform is added to .the serum and the mixture is shaken for 3 hours at 25 C. The mixture is allowed to stand at room temperature for several hours and then is centrifuged at 7,000 X g for 10 minutes whereupon three layers form, a solvent layer, a layer containing a solid precipitate and a serum liquid layer. The three layers are separated and the serum layer is placed under vacuum at 35 with stirring (for at least 15' minutes) until foaming ceases. The pH of the serum is adjusted to 7.2 by adding 5N sodium hydroxide thereto. About 1 g kaolin is added to the serum and the mixture isstirred on a magnetic stirrer for 1% hours. The serum is then filtered through a sterile D-8 filterer pad to thereby remove additional impurities therefrom.

EXAMPLE 4 The pH of human serum (plasma with substantially all fibrinogen removed therefrom) is adjusted to a value of 5.4 by adding about 1 ml of dilute acetic acid (30 percent), to 100 ml of serum. The serum is shaken for 3 hours at a temperature of about 35 C. and the resulting precipitate removed. About 300 ml of chloroform is mixed with the supernatent and the mixture is allowed to stand at room temperature for several hours and then is centrifuged at 7,000 X g for 10 minutes whereupon three layers form, a solvent layer, a layer containing a solid precipitate and a serum liquid layer. The three layers are separated and the serum layer is placed under vacuum at 35 with stirring (for at least minutes) until foaming ceases. The pH is adjusted to' 7.2 by adding 5N sodium hydroxide thereto. About 1 g kaolin is added to the serum and the mixture is stirred on a magnetic stirrer for 1% hours. The serum is then filtered through a sterile D-8 filter pad to thereby remove additional impurities therefrom.

The treated human serum obtained in each of examples l to 4 is substantially free of particle-forming protein and is acceptable for grouping and Rh typing purpose. Furthermore, the antibody titre of the serum is not significantly affected by the procedure described in these examples.

What is claimed is:

1. A process for substantially preventing or retarding the formation of particles in blood sera induced by shaking, by removal of particle-forming proteins therefrom, which comprises adjusting pH of the blood serum to within the range of from about 5.2 to about 5.6, mixing or extracting the blood serum at ambient temperature up to about 35 C with from about 0.5 to about 5 volumes of a halogenated straight chain hydrocarbon per volume of blood serum, thereby causing the formation of a proteinaceous precipitate, and separating the precipitate from the serum.

2. A process in accordance with claim 1 wherein the extraction of the serum is carried out at a temperature within the range of from about 20 to about 35 C. v

3. A process in accordance with claim 2 wherein the extraction is carried out at ambient t rnperature.

4. A process In accordance with c arm 1 wherein the pH employed is within the range of from about 5.2 to about 5 .4.

5. A process in accordance with claim 1 wherein the halogenated straight chain hydrocarbon is chloroform.

6. A process in accordance with claim 1 wherein the halogenated straight chain hydrocarbon is methylene chloride.

7. A process in accordance with claim 1 wherein the halogenated straight chain hydrocarbon is carbon tetrachloride.

8. A process in accordance with claim 1 wherein the serum adjusted to a pH within the range of from about 5.2 to about 5.6 is shaken to induce precipitation of proteinaceous particles and after removing the precipitate, the resulting supernatent is mixed with the halogenated straight chain hydrocarbon. 

2. A process in accordance with claim 1 wherein the extraction of the serum is carried out at a temperature within the range of from about 20* to about 35* C.
 3. A process in accordance with claim 2 wherein the extraction is carried out at ambient temperature.
 4. A process in accordance with claim 1 wherein the pH employed is within the range of from about 5.2 to about 5.4.
 5. A process in accordance with claim 1 wherein the halogenated straight chain hydrocarbon is chloroform.
 6. A process in accordance with claim 1 wherein the halogenated straight chain hydrocarbon is methylene chloride.
 7. A process in accordance with claim 1 wherein the halogenated straight chain hydrocarbon is carbon tetrachloride.
 8. A process in accordance with claim 1 wherein the serum adjusted to a pH within the range of from about 5.2 to about 5.6 is shaken to induce precipitation of proteinaceous particles and after removing the precipitate, the resulting supernatent is mixed with the halogenated straight chain hydrocarbon. 